Metabolites In Drug Development Research Articles

Revealing The Motifs, Properties, and Phylogeny of Lupeol Synthase Using Bioinformatics Approach

Lupane-type triterpenoid saponins are potent plants’ secondary metabolites for drug development as they showed various activities such as anticancer, Sarcoplasmic Reticulum Ca 2+ -ATPase (SERCA) activator which is very important for neurons, and antileishmanial. The triterpenoid saponin backbone is produced by cyclization and rearrangement of the 2,3-oxidosqualene precursor by the oxidosqualene cyclase. The type of oxidosqualene cyclase involved determines the type of saponins so it is referred to as a key enzyme. Lupane-type saponins are produced by 2,3-oxidosqualene cyclization through the chair-chair-chair conformation and the formation of various cation intermediates. This study aimed to analyze lupeol synthase, the key enzyme which determines the conversion of 2,3-oxidosqualene into lupane-type saponins. This in silico project was done using bioinformatics programs including Multiple Em for Motif Elicitation (MEME), ProtParam, and Molecular Evolutionary Genetic Analysis (MEGA-X) for relationship analysis. The amino acid sequences analysis using the MEME program showed that lupeol synthase has QW, DCTAE, and CYCR conserved motifs in the oxidosqualene cyclase family even though some evolutions were also present. Analysis of chemical and physical parameters with ProtParam indicated that lupeol synthase had lower stability than lanosterol synthase from Saccharomyces cerevisiae . The phylogenetic tree showed that lupeol synthase was closely related to other plant oxidosqualene cyclases. The results of this study are expected to support the modification strategy determination to increase the production of lupane-type saponins using a biotechnological approach in the pharmaceutical industry.

Potential of kynurenine metabolites in drug development against neurodegenerative diseases.

Neural Regen Res, Official publication of Publishing House of Neural Regeneration Research China Association of Rehabilitation Medicine,China

Antidiabetic Potential of Marine Brown Algae-a Mini Review.

Marine algae are an important source of bioactive metabolites in drug development and nutraceuticals. Diabetes mellitus is a metabolic disorder and the third leading cause of death worldwide due to lifestyle changes associated with rapid urbanization. Due to the adverse side effects of currently available antidiabetic drugs, search for an effective natural-based antidiabetic drug is important to combat diabetes and its complications. Therefore, in lieu with herbal drug development, it is important to find the potential benefits of seaweeds for the management of type 2 diabetes as they are underexplored yet in Sri Lanka. Among the marine seaweeds, natural bioactive compounds are abundant in brown algae with potentials in application as active ingredients in drug leads and nutraceuticals. Bioactive secondary metabolites are derived from numerous biosynthetic pathways of marine algae which contribute to various chemical and biological properties. Phlorotannins present in marine brown algae exhibited antidiabetic activities through different mechanisms such as the inhibitory effect of enzyme targets mainly by inhibiting the enzymes such as α-amylase, α-glucosidase, angiotensin-converting enzymes (ACE), aldose reductase, dipeptidyl peptidase-4, and protein tyrosine phosphatase 1B (PTP 1B) enzyme. In addition, phlorotannins derived from brown algae have the ability to reduce diabetic complications. Hence, the present review focuses on the different antidiabetic mechanisms of secondary bioactive compounds present in marine brown algae.

A Decade in the MIST: Learnings from Investigations of Drug Metabolites in Drug Development under the "Metabolites in Safety Testing" Regulatory Guidance.

Since the introduction of metabolites in safety testing (MIST) guidance by the Food and Drug Administration in 2008, major changes have occurred in the experimental methods for the identification and quantification of metabolites, ways to evaluate coverage of metabolites, and the timing of critical clinical and nonclinical studies to generate this information. In this cross-industry review, we discuss how the increased focus on human drug metabolites and their potential contribution to safety and drug-drug interactions has influenced the approaches taken by industry for the identification and quantitation of human drug metabolites. Before the MIST guidance was issued, the method of choice for generating comprehensive metabolite profile was radio chromatography. The MIST guidance increased the focus on human drug metabolites and their potential contribution to safety and drug-drug interactions and led to changes in the practices of drug metabolism scientists. In addition, the guidance suggested that human metabolism studies should also be accelerated, which has led to more frequent determination of human metabolite profiles from multiple ascending-dose clinical studies. Generating a comprehensive and quantitative profile of human metabolites has become a more urgent task. Together with technological advances, these events have led to a general shift of focus toward earlier human metabolism studies using high-resolution mass spectrometry and to a reduction in animal radiolabel absorption/distribution/metabolism/excretion studies. The changes induced by the MIST guidance are highlighted by six case studies included herein, reflecting different stages of implementation of the MIST guidance within the pharmaceutical industry.

Quantitative Prediction of Drug-Drug Interactions Involving Inhibitory Metabolites in Drug Development: How Can Physiologically Based Pharmacokinetic Modeling Help?

This subteam under the Drug Metabolism Leadership Group (Innovation and Quality Consortium) investigated the quantitative role of circulating inhibitory metabolites in drug–drug interactions using physiologically based pharmacokinetic (PBPK) modeling. Three drugs with major circulating inhibitory metabolites (amiodarone, gemfibrozil, and sertraline) were systematically evaluated in addition to the literature review of recent examples. The application of PBPK modeling in drug interactions by inhibitory parent–metabolite pairs is described and guidance on strategic application is provided.

An Industry Perspective on Tiered Approach to The Investigation of Metabolites in Drug Development

A tiered approach to drug metabolite measurement and identification is often used industry wide to fulfill regulatory requirements specified in recent US FDA and European Medicines Agency guidance. Although this strategy is structured in its intent it can be customized to address unique challenges which may arise during early and late drug development activities. These unconventional methods can be applied at any stage to facilitate metabolite characterization. Two case studies are described NVS 1 and 2. NVS 1: plasma concentrations, measured using a radiolabeled MS-response factor exploratory method, were comparable to those from a validated bioanalytical method. The NVS 2 example showed how in vitro analysis helped to characterize an unexpectedly abundant circulating plasma metabolite M3. A tiered approach incorporating many aspects of conventional and flexible analytical methodologies can be pulled together to address regulatory questions surrounding drug metabolite characterization.

Data-Driven Approach for Cross-Species Comparative Metabolite Exposure Assessment: How to Establish Fundamental Bioanalytical Parameters for The Peak Area Ratio Method

We previously developed an high-performance LC-MS peak area ratio approach to demonstrate whether an animal species used in a toxicology study has greater exposures to drug metabolites relative to humans, meeting regulatory guidances regarding safety assessment of drug metabolites. Herein we explain the underlying bioanalytical principals, how to establish all fundamental bioanalytical parameters, and how to evaluate data quality in sample analysis, in the absence of authentic standards of analyte(s). A data-driven tiered approach was used in which data from the peak area ratio method can stand based on statistical analysis, as well as assuring that fundamental elements of bioanalytical method and bioanalysis are met. This strategy offers considerable time- and resource-saving advantage while providing high confidence in the safety assessment of human metabolites in drug development.

Managing the challenge of chemically reactive metabolites in drug development

The normal metabolism of drugs can generate metabolites that have intrinsic chemical reactivity towards cellular molecules, and therefore have the potential to alter biological function and initiate serious adverse drug reactions. Here, we present an assessment of the current approaches used for the evaluation of chemically reactive metabolites. We also describe how these approaches are being used within the pharmaceutical industry to assess and minimize the potential of drug candidates to cause toxicity. At early stages of drug discovery, iteration between medicinal chemistry and drug metabolism can eliminate perceived reactive metabolite-mediated chemical liabilities without compromising pharmacological activity or the need for extensive safety evaluation beyond standard practices. In the future, reactive metabolite evaluation may also be useful during clinical development for improving clinical risk assessment and risk management. Currently, there remains a huge gap in our understanding of the basic mechanisms that underlie chemical stress-mediated adverse reactions in humans. This review summarizes our views on this complex topic, and includes insights into practices considered by the pharmaceutical industry.

Immunostimulatory effects of Uncaria perrottetii (A. Rich.) Merr. (Rubiaceae) vinebark aqueous extract in Balb/C mice

Crude extract of Uncaria perrottetii (A. Rich.) Merr. vinebark was evaluated for its immunomodulating activity in Balb/C mice. Initially, the immunomodulatory potential of the plant extract was evaluated using in vitro immune response assays at different concentrations of the plant extract (10 μg/mL, 20 μg/mL, 50 μg/mL and 100 μg/mL). Using the optimum concentration determined in the in vitro assays, the protective effect of the plant extract was assessed against drug-induced immunosuppression in vivo. For the in vivo experiment, thirty-six (36) mice were divided into 3 groups of 12 mice each: (1) cyclophosphamide drug-induced (30 mg/kg BW) immunosuppressed mice (Cy group) served as the positive control; (2) Uncaria perrottetii extract and Cy-treated mice (U+Cy); and (3) PBS-injected mice as the negative control group [(-) CTRL]. The optimum concentration was determined to be 50 μg/mL in the in vitro assays. At this concentration, Uncaria perrottetii extract stimulated peritoneal phagocyte activation, produced a significant increase in the activity of phagocytic cells from the spleen and promoted splenic cellular proliferation with or without lipopolysaccharide (LPS) when compared with the PBS-treated cells (negative control). Moreover, cells treated with 50 μg/mL of Uncaria perrottetii increased macrophage respiratory burst activity that was comparable to that of the phorbol myristate acetate-stimulated splenic macrophages. In all immune assays undertaken in the in vivo experiment, the Cy-treated mice showed significantly lower response when compared with the PBS-treated mice. Significant improvement in peritoneal cell activation, phagocytic activity and cellular proliferation was exhibited by the U+Cy-treated mice when compared with Cy-injected mice. The extract from Uncaria perrottetii also significantly enhanced respiratory burst and plasma lysozyme activity compared with the Cy-injected mice. Based on the results of both in vitro and in vivo trials, Uncaria perrottetii extract has immunopotentiating activities on the innate immunity of Balb/C mice and the extract could potentially reverse the immunosuppressive effects of Cy. However, the potential of the plant as source of bioactive products and metabolites for drug development still has to be fully investigated.

Generation and Identification of Reactive Metabolites by Electrochemistry and Immobilized Enzymes Coupled On-Line to Liquid Chromatography/Mass Spectrometry

The detection of reactive metabolites using conventional in vivo and in vitro techniques is hampered because the intermediately formed reactive species are prone to covalent binding to cellular macromolecules. Therefore, the application of improved methods is required. The on-line coupling of an electrochemical reactor and horseradish peroxidase immobilized on magnetic microparticles with liquid chromatography/mass spectrometry (EC/LC/MS or HRP/LC/MS) allows the direct detection of reactive metabolites of the model compounds amodiaquine, amsacrine, and mitoxantrone, which are all known for readily binding to cellular macromolecules after metabolization by cytochrome P450. EC/LC/MS and HRP/LC/MS experiments were compared to rat liver microsome incubations and proved to be valuable complementary methods since reactive quinone, quinone imine, and quinone diimine species could be detected directly and not only after trapping with glutathione. Furthermore, N-dealkylation and N-oxidation of amodiaquine were successfully simulated by electrochemical oxidation reactions, as well as the formation of an aldehyde. Therefore, EC/LC/MS and HRP/LC/MS are promising tools for the identification of both reactive and stable metabolites in drug development.

Active drug metabolites in drug development.

Active drug metabolites discovered during the course of drug development constitute a subset of the larger issue of metabolic drug interactions, but still demand unique consideration from both an efficacy and a safety point of view. Improved technology has allowed better identification and quantification of metabolites, raising new issues to be addressed during the course of drug development. Several new molecular identities recently entering the marketplace have active metabolites, and many more are (or have been) in development. The MIST (Metabolites in Safety Testing) committee of PhRMA (Pharmaceutical Research and Manufacturers of America) has prepared a position paper (in press) on the subject, which has been widely discussed (with and by regulatory authorities) over the past three years.